DNA methyltransferase mediates dose-dependent stimulation of neural stem cell proliferation by folate

The proliferative response of neural stem cells (NSCs) to folate may play a critical role in the development, function and repair of the central nervous system. It is important to determine the dose-dependent effects of folate in NSC cultures that are potential sources of transplantable cells for therapies for neurodegenerative diseases. To determine the optimal concentration and mechanism of action of folate for stimulation of NSC proliferation in vitro, NSCs were exposed to folic acid or 5-methyltetrahydrofolate (5-MTHF) (0–200 μmol/L) for 24, 48 or 72 h. Immunocytochemistry and methyl thiazolyl tetrazolium assay showed that the optimal concentration of folic acid for NSC proliferation was 20–40 μmol/L. Stimulation of NSC proliferation by folic acid was associated with DNA methyltransferase (DNMT) activation and was attenuated by the DNMT inhibitor zebularine, which implies that folate dose-dependently stimulates NSC proliferation through a DNMT-dependent mechanism. Based on these new findings and previously published evidence, we have identified a mechanism by which folate stimulates NSC growth.     

Down-expressed GLT-1 in PSD astrocytes inhibits synaptic formation of NSC-derived neurons in vitro

Little is known about the effect of astroglial GLT-1 of post-stroke depression (PSD) rat model on the function of neural stem cells (NSCs). This study aimed to investigate whether astroglial GLT-1 of PSD rats affect differentiation of NSCs from neonatal rat hippocampus and synaptic formation of NSC-derived neurons. Astrocytes were isolated from the left hippocampus of normal adult SD rats and PSD rats. A lentiviral vector was used to silence the expression of GLT-1 in astrocytes of PSD rats. NSCs were respectively co-cultured with normal (control), PSD, and GLT-1 silenced astrocytes for 7 days. GLT-1, GFAP, MAP2, Synaptophysin (SYN), glutamate (Glu) and glutamine (Gln) were respectively measured by qRT-PCR, western blot, immunofluorescence and efficient mass spectrometry (MS). PSD astrocytes increased the number of NSC-derived astrocytes, but inhibited the expression of GLT-1 of NSC-derived astrocytes and synapses of NSC-derived neurons. On the basis of the low expression of GLT-1 in PSD astrocytes, we further silenced GLT-1 in PSD astrocytes. Interestingly, GLT-1 silenced PSD astrocytes more obviously inhibited synapses of NSC-derived neurons, but increased the number of NSC-derived neurons and reversed the expression of GLT-1 in NSC-derived astrocytes. At the same time, concentration of glutamate in the medium elevated, and glutamine in the medium gradually reduced. In NSC-derived neurons and astrocytes, glutamate metabolism was also affected by changed GLT-1. Down-expressed GLT-1 in PSD astrocytes stimulated NSCs differentiating into astrocytes, but inhibiting the formation of functional synapses by influencing glutamate metabolism in vitro.     

Rosmarinic Acid Ameliorates Depressive-Like Behaviors in a Rat Model of CUS and Up-Regulates BDNF Levels in the Hippocampus and Hippocampal-Derived Astrocytes

Rosmarinic acid (RA), a primary constituent of a Chinese herbal medicine, has been shown to have some therapeutic effects in an animal model of depression, but its underlying mechanisms are poorly understood. Sprague–Dawley rats were exposed to chronic unpredictable stress (CUS) for 21 days, and received RA for 14 days from the last week of CUS, then the behavioral changes, hippocampal pERK1/2 and BDNF levels were observed. Rats were further treated with U0126 (an ERK1/2 phosphorylation inhibitor) 30 min before RA treatment to assess the effects of RA and ERK1/2 signaling in depressive-like behavior and hippocampal BDNF levels. In addition, brains of newly born Sprague–Dawley rats were used to harvest and expand hippocampal astrocytes. Cells were exposed to different concentrations of RA (sham, 1, 5, 10, 20, and 40 μg/mL) or U0126 (2 μM as a final concentration) + RA (sham, 1, 5, 10, 20, and 40 μg/mL) for 48 h, and the pERK1/2 and BDNF levels were assessed by western and ELISA assays. RA administration (10 mg/kg daily) reversed depressive-like behaviors in rats exposed to a chronic unpredictable stress paradigm and restored pERK1/2 protein expression and hippocampal brain-derived neurotrophic factor (BDNF). Moreover, in vitro experiments revealed that 20 μg/mL RA increased pERK1/2 and BDNF levels in cultured astrocytes. Interestingly, the effects of RA were inhibited by U0126. RA might be a useful treatment for depression and the changes in ERK1/2 signaling and BDNF levels may play a critical role in the pharmacological action of RA.     

一氧化氮促进体外培养大鼠脑室下区神经干细胞的分化

目的：探讨一氧化氮（NO）对新生大鼠体外培养的神经干细胞（NSCs）分化的作用。方法：采用常规方法分离新生大鼠脑室下区（SVZ）组织,进行NSCs体外培养。用DETA／NO作为NO供体,用L-NAME作为一氧化氮合酶（NOS）抑制剂。免疫荧光法检测NSCs标志物-巢蛋白（nestin）、神经元标志物-8Ⅲ型微管蛋白（Tuj-1）和星型胶质细胞标志物-胶质原纤维酸性蛋白（GFAP）的表达,还检测了神经元型NOS的表达。用Greiss还原法检测培养液中总NO的浓度。结果：培养的神经球均为nestin阳性、BIdu阳性和nNOS阳性。NSCs和40μmol／L、50μmol／L、60μmol／LDEFA／N0共培养5d,实验组培养液中N0浓度较对照组显著增高（P〈0．01）,相应实验组分化的神经元数和星型胶质细胞数较对照组明显增加（P〈0．01和P〈0．05）。NSCs和100μmol／L、150μmol／L、200μmol／LL-NAME共培养5d,实验组培养液中NO浓度较对照组降低（P〈0．05）,相应实验组分化的神经元数和星型胶质细胞数也较对照组减少（P〈0．05）。结论：NO能直接促进大鼠SVZ体外培养的NSCs分化。     

Effects of Homocysteine on ERK Signaling and Cell Proliferation in Fetal Neural Stem Cells In Vitro

The aim of the present study was to determine if the excitatory amino acid homocysteine (Hcy) alters ERK signaling and cell proliferation in fetal neural stem cells (NSCs) in vitro. NSCs were isolated from fetal rats and grown in serum-free suspension medium. The cells were identified as NSCs by their expression of immunoreactive Sox2. NSCs were assigned to one of four treatment groups: vehicle control, low-dose Hcy group (Hcy-L, medium contained 30 μmol/L Hcy), middle-dose Hcy group (Hcy-M, 100 μmol/L Hcy) and high-dose Hcy group (Hcy-H, 300 μmol/L Hcy). Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Protein expression levels of ERK1/2 and phosphorylated ERK1/2 were detected by Western blot. The effects of Hcy on NSC death, including apoptosis, were assessed by using flow cytometry and trypan blue exclusion. The results showed that NSCs grew as neurospheres in the serum-free medium. Hcy decreased ERK1/2 protein phosphorylation and NSC proliferation, but it did not induce cell death or apoptosis within the concentration from 30 to 300 μmol/L. The above results are consistent with the hypothesis that Hcy decreases fetal NSC proliferation by inhibiting ERK signaling.     

RCMV感染星形胶质细胞对神经干细胞分化的影响

探讨大鼠巨细胞病毒（rat cytomegalovirus,RCMV）感染大鼠星形胶质细胞后,对神经干细胞分化的影响。原代分离培养新生大鼠星形胶质细胞和胚胎海马神经干细胞,将星形胶质细胞感染RCMV后和神经干细胞在Transwell24孔共培养体系下进行共培养,同时设对照组;用免疫荧光染色等方法检测神经干细胞与感染RCMV的星形胶质细胞共培养后,其分化细胞中神经元微管相关蛋白（microtubule-associated protein 2,MAP2）和星形胶质细胞胶质纤维酸性蛋白（glial fibril—lary acidic protein,GFAP）的表达。结果发现,感染RCMV的星形胶质细胞与神经干细胞共培养时,神经干细胞分化减慢,分化成的神经元和星形胶质细胞比率低于对照组,提示星形胶质细胞感染RCMV后可抑制神经干细胞的分化,可能与RCMV影响星形胶质细胞合成和分泌各种营养因子,干扰了神经干细胞的分化进程有关。     

肿瘤坏死因子-α对大鼠中脑神经干细胞分化和寡突胶质细胞增殖的影响

为观察肿瘤坏死因子对神经干细胞(NSCs)分化的影响,本研究应用体外扩增的新生大鼠中脑NSCs,使用免疫组织化学技术,观察了肿瘤坏死因子—α(TNF—α)对NSCs分化及其后代细胞的影响。结果显示：(1)TNF—α可提高中脑NSCs后代中神经元和寡突胶质细胞所占的比例;(2)TNF—α可明显诱导由NSCs分化的寡突胶质细胞增殖,但对星形胶质细胞的增殖作用不明显。上述观察结果提示TNF—α对NSCs的应用具有重要影响。     

Antileukemic activity of combined epigenetic agents, DNMT inhibitors zebularine and RG108 with HDAC inhibitors, against promyelocytic leukemia HL-60 cells

DNMT inhibitors are promising new drugs for cancer therapies. In this study, we have observed the antileukemic action of two diverse DNMT inhibitors, the nucleoside agent zebularine and the non-nucleoside agent RG108, in human promyelocytic leukemia (PML) HL-60 cells. Zebularine but not RG108 caused dose- and time-dependent cell growth inhibition and induction of apoptosis. However, co-treatment with either drug at a non-toxic dose and all trans retinoic acid (RA) reinforced differentiation to granulocytes, while 24 or 48 h-pretreatment with zebularine or RG108 followed by RA alone or in the presence of HDAC inhibitors (sodium phenyl butyrate or BML-210) significantly accelerated and enhanced cell maturation to granulocytes. This occurs in parallel with the expression of a surface biomarker, CD11b, and early changes in histone H4 acetylation and histone H3K4me3 methylation. The application of both drugs to HL-60 cells in continuous or sequential fashion decreased DNMT1 expression, and induced E-cadherin promoter demethylation and reactivation at both the mRNA and the protein levels in association with the induction of granulocytic differentiation. The results confirmed the utility of zebularine and RG108 in combinations with RA and HDAC inhibitors to reinforce differentiation effects in promyelocytic leukemia.     

Effects of ginkgoneolic acid on the growth, acidogenicity, adherence, and biofilm of Streptococcus mutans in vitro

Ginkgo biloba has long been used in traditional Chinese medicine. In this study, ginkgoneolic acid, a kind of compound extracted from G. biloba, was investigated for its effects on growth, acid production, adherence, biofilm formation, and biofilm morphology of Streptococcus mutans. The results showed that ginkgoneolic acid inhibited not only the growth of S. mutans planktonic cells at minimum inhibitory concentration (MIC) of 4 μg/mL and minimum bactericidal concentration (MBC) of 8 μg/mL but also the acid production and adherence to saliva-coated hydroxyapatite of S. mutans at sub-MIC concentration. In addition, this agent was effective in inhibiting the biofilm formation of S. mutans (MBIC₅₀?=?4 μg/mL), and it reduced 1-day-developed biofilm of S. mutans by 50 % or more at low concentration (MBRC₅₀?=?32 μg/mL). Furthermore, the present study demonstrated that ginkgoneolic acid disrupted biofilm integrity effectively. These findings suggest that ginkgoneolic acid is a natural anticariogenic agent in that it exhibits antimicrobial activity against S. mutans and suppresses the specific virulence factors associated with its cariogenicity.     

Gastrodin Ameliorates Depressive-Like Behaviors and Up-Regulates the Expression of BDNF in the Hippocampus and Hippocampal-Derived Astrocyte of Rats

Gastrodin (GAS), a main constituent of a Chinese herbal medicine Tian ma, has been shown to be effective in treating various mood disorders. The purpose of the present study was to assess the effects of GAS on alleviating depressive-like behaviors in a rat model of chronic unpredictable stress (CUS) and regulating the expression of BDNF in the hippocampus and hippocampal-derived astrocyte from Sprague–Dawley (SD) rats. Following CUS, rats were intraperitoneally administered gastrodin (50, 100, or 200 mg/kg daily) or vehicle for 2 weeks. Rats were then experienced sucrose preference test and forced swim test. The expressions of GFAP and BDNF in the hippocampus were evaluated. In addition, hippocampal astrocytes were isolated from neonatal SD rats and exposed to different concentrations of GAS (sham, 5, 10, 20, 50 and 100 μg/mL) for 48 and 72 h before the cell viability and the levels of pERK1/2 and BDNF were analyzed. Furthermore, the cell viability was also tested after exposure to serum-free condition that contain different concentrations of GAS for 48 and 72 h. GAS administration (100 and 200 mg/kg daily) reversed depressive-like behaviors in rats exposed to CUS paradigm and restored the expression of GFAP and BDNF in the hippocampus. Moreover, in vitro experiments revealed that GAS did not increase the cell viability of astrocytes but protected it from 72 h’s serum-free damage at the dosage 20 μg/mL. Increased levels of ERK1/2 phosphorylation and BDNF protein were also observed after GAS (20 μg/mL) treatment for 72 h. These results indicate that gastrodin possesses antidepressant effect. The changes of the astrocyte activation and the level of BDNF may play a critical role in the pharmacological action of GAS.     

Lactoferrin promote primary rat osteoblast proliferation and differentiation via up-regulation of insulin-like growth factor-1 expression

The aim of this study was to explore the effect of lactoferrin (LF) in primary fetal rat osteoblasts proliferation and differentiation and investigate the underlying molecular mechanisms. Primary rat osteoblasts were obtained from the calvarias of neonatal rats. Osteoblasts were treated with LF (0.1–1000 μg/mL), or OSI-906 [a selective inhibitor of insulin-like growth factor 1 (IGF-1) receptor and insulin receptor]. The IGF-1 was then knocked down by small hairpin RNA (shRNA) technology and then was treated with recombinant human IGF-1 or LF. Cell proliferation and differentiation were measured by MTT assay and alkaline phosphatase (ALP) assay, respectively. The expression of IGF-1 and IGF binding protein 2 (IGFBP2) mRNA were analyzed using real-time PCR. LF promotes the proliferation and differentiation of osteoblasts in a certain range (1–100 μg/mL) in time- and dose-dependent manner. The mRNA level of IGF-1 was significantly increased, while the expression of IGFBP2 was suppressed by LF treatment. Knockdown of IGF-1 by shRNA in primary rat osteoblast dramatically decreased the abilities of proliferation and differentiation of osteoblasts and blocked the proliferation and differentiation effect of LF in osteoblasts. OSI906 (5 μM) blocked the mitogenic and differentiation of LF in osteoblasts. Proliferation and differentiation of primary rat osteoblasts in response to LF are mediated in part by stimulating of IGF-1 gene expression and alterations in the gene expression of IGFBP2.     

In vitro and in silico characterization of angiogenic inhibitors from <Emphasis Type="Italic">Sophora interrupta</Emphasis>

Sophora interrupta Bedd, (Fabaceae) is used in Indian folk medicine to treat cancer. Angiogenesis is one of the crucial characteristics of cancer metastasis and is regulated by vascular endothelial growth factor (VEGF). In this study, we examined the antiangiogenic properties of the root ethyl acetate extract of Sophora interrupta by various methods. In vitro antioxidant activity (100–600 μg/ml) of S. interrupta ethyl acetate (SEA) extract was evaluated by DPPH and ABTS, anti-inflammatory activity (50, 100 and 150 μg/ml) by estimating nitric oxide (NO) levels, anti-angiogenic activity (200 and 500 μg/ml) was validated by chorio allantoic membrane (CAM) assay and in silico molecular dynamic (MD) simulations analyses (25 ns) were performed to identify the anti-angiogenic compounds extracted from root extract. The antioxidative activity of SEA extract at IC₅₀ (200?±?0.6 μg/mL) is equal to that of ascorbic acid at IC₅₀ (50?±?0.6 μg/mL), and the anti-inflammatory activity of SEA extract at IC₅₀ (150?±?0.2 μg/mL) was inhibited significantly by nitric oxide (NO) production. The SEA extract significantly reduced the sprouting of new blood vessels at ID₅₀ 500?±?0.13 μg/mL in the CAM assay. Gas chromatography–mass spectrometry analysis of the SEA extract detected 34 secondary metabolites, of which 6a,12a-dihydro-6H-(1,3)dioxolo(5,6)benzofuro(3,2-c)chromen-3-ol (maackiain) and funiculosin formed strong hydrogen bond interactions with Lys 920, Thr 916 and Cys 919 (2H), as well as Glu 917 of VEGFR2, and these interactions were similar to those of the anti-angiogenic compound axitinib. Significant findings in all the assays performed indicate that SEA extract has potential anti-angiogenic compounds that may interfere with VEGF-induced cancer malignancy.     

Particulate Matter Facilitates C6 Glioma Cells Activation and the Release of Inflammatory Factors Through MAPK and JAK2/STAT3 Pathways

It has been widely accepted that astrocytes, play a role in regulating almost every physiological system. In the present study, we investigated the role of particulate matter (PM) in regulating activation of astrocytes. The glial cell strain C6 was cloned from a rat glioma which was induced by N-nitrosomethylurea. The C6 cells were plated at a density of 5 × 10⁶ cells/10 cm diameter dish and incubated with different concentrations (0, 12, 25, 50, 100, 200, and 400 μg/mL) of PM for 24 h and different time (0, 1, 3, 6, 8,12, and 24 h) with 100 μg/mL at 37 °C. The study revealed that PM stimulated the expression of inducible nitric oxide synthase (iNOS) as well as the production of IL-1β in a dose- and time-dependent manner. Furthermore, activation of JAK2/STAT3 and p38/JNK/ERK MAPKs was found in astrocytes following PM treatment. Blockage of JAK and p38/JNK/ERK MAPKs with their specific inhibitors, AG490, SB202190, SP600125 and U0126 significantly reduced PM-induced iNOS expression and IL-1β production. In addition, it was demonstrated that inhibition of p38, JNK and JAK prevented STAT3 tyrosine phosphorylation induced by PM, while blocking ERK did not. MAPKs (p38 and JNK) could regulate tyrosine STAT3 phosphorylation, which suggested that the JAK2/STAT3 pathway might be the downstream of p38/JNK MAPK pathways.     

The effect of glutamate-induced excitotoxicity on DNA methylation in astrocytes in a new in vitro neuron-astrocyte-endothelium co-culture system

Glutamate-induced excitotoxicity is a contributer to many neurological diseases. Astrocytes may represent a new target for treating glutamate-induced excitotoxicity. However, the in vitro culture system that mimics the in vivo microenvironment is lacking. This study aimed to establish a new in vitro co-culture system including neurons, astrocytes, and endothelial cells (NAE), and to investigate the effect of glutamate-induced excitotoxicity on DNA methylation in astrocytes. A NAE co-culture method was created using a Transwell chamber, in which neurons were seeded on the bottom of the lower chamber, endothelial cells were plated on the top membrane, and astrocytes were plated on the bottom membrane of the insert. Glutamate-induced toxicity was induced using glutamate and glycine, and examined using immunofluorescence and lactate dehydrogenase release assay. Global methylation in astrocytes was analyzed, and the expression of DNMT1 and DNMT3a was examined using Western blot analysis. Glutamate treatment induced less neuronal damage in the NAE system compared with the control group in which neurons and astrocytes were cultured alone. Global DNA methylation was increased and the expression of DNMT1 and DNMT3a in astrocytes was increased after glutamate treatment, which was blocked by application of the NMDAR inhibitor MK-801 and the DNMT inhibitor 5-azaC from the endothelial cells. The in vitro ANE culture system is effective for studying glutamate-induced excitotoxicity, and may be used for testing the passage of drugs across the blood–brain barrier. Inhibition of DNA methylation in astrocytes may be a new therapeutic strategy for treating glutamate-induced excitotoxicity.     

Beta 1 integrin-mediated effects of tenascin-R domains EGFL and FN6-8 on neural stem/progenitor cell proliferation and differentiation in vitro

Neural stem/progenitor cells (NSCs) have the capacity for self-renewal and differentiation into major classes of central nervous system cell types, such as neurons, astrocytes, and oligodendrocytes. The determination of fate of NSCs appears to be regulated by both intrinsic and extrinsic factors. Mounting evidence has shown that extracellular matrix molecules contribute to NSC proliferation and differentiation as extrinsic factors. Here we explore the effects of the epidermal growth factor-like (EGFL) and fibronectin type III homologous domains 6-8 (FN6-8) of the extracellular matrix molecule tenascin-R on NSC proliferation and differentiation. Our results show that domain FN6-8 inhibited NSC proliferation and promoted NSCs differentiation into astrocytes and less into oligodendrocytes or neurons. The EGFL domain did not affect NSC proliferation, but promoted NSC differentiation into neurons and reduced NSC differentiation into astrocytes and oligodendrocytes. Treatment of NSCs with beta 1 integrin function-blocking antibody resulted in attenuation of inhibition of the effect of FN6-8 on NSC proliferation. The influence of EGFL or FN6-8 on NSCs differentiation was inhibited by beta 1 integrin antibody application, implicating beta 1 integrin in proliferation and differentiation induced by EGFL and FN6-8 mediated triggering of NSCs.     

胎鼠脊髓源性神经干细胞分离培养与鉴定

目的:研究胎鼠的脊髓源性神经干细胞的分离培养方法并观察其增殖和分化能力。方法:利用显微操作技术分离获得胎鼠脊髓组织、无血清培养技术和酶消化法结合机械法传代培养神经干细胞、免疫细胞化学方法鉴定神经干细胞和分化情况。结果:建立了胎鼠脊髓源性神经干细胞的分离、培养和鉴定的方法,观察到了脊髓源性神经干细胞具有较强的增殖能力,在添加有5ng／mlEGF和5ng／mlbFGF的无血清培养液中可贴壁分化为神经元、少突细胞和星形胶质细胞。结论:在体外培养条件下分离培养的胎鼠脊髓源性神经干细胞具有干细胞的特性即较强的增殖能力和多向分化潜能。     

The in vitro genotoxicity of benzoic acid in human peripheral blood lymphocytes

The chromosomal aberration (CA), sister chromatid exchange (SCE) and micronucleus test (MN) were employed to investigate the in vitro effect of antimicrobial food additive benzoic acid on human chromosomes. Lymphocytes were incubated with various concentrations (50, 100, 200 and 500 μg/mL) of benzoic acid. The results of used assays showed that benzoic acid significantly increased the chromosomal aberration, sister chromatid exchange and micronucleus frequency (200 and 500 μg/mL) without changing the pH of the medium in a dose-dependent manner. Also this additive significantly decreased the mitotic index (MI) at the highest concentration for 24 h and 100, 200 and 500 μg/mL for 48 h. This decrease was dose-dependent as well. However, it did not effect the replication (RI) and nuclear division (NDI) indices.     

In vitro characterization of subventricular zone isolated neural stem cells,from adult monkey and rat brain

Neural stem cells (NSCs) are multipotent, self-renewable cells who are capable of differentiating into neurons, astrocytes, and oligodendrocytes. NSCs reside at the subventricular zone (SVZ) of the adult brain permanently to guarantee a lifelong neurogenesis during neural network plasticity or undesirable injuries. Although the specious inaccessibility of adult NSCs niche hampers their in vivo identification, researchers have been seeking ways to optimize adult NSCs isolation, expansion, and differentiation, in vitro. NSCs were isolated from rhesus monkey SVZ, expanded in vitro and then characterized for NSCs-specific markers expression by immunostaining, real-time PCR, flow cytometry, and cell differentiation assessments. Moreover, cell survival as well as self-renewal capacity were evaluated by TUNEL, Live/Dead and colony assays, respectively. In the next step, to validate SVZ-NSCs identity in other species, a similar protocol was applied to isolate NSCs from adult rat’s SVZ as well. Our findings revealed that isolated SVZ-NSCs from both monkey and rat preserve proliferation capacity in at least nine passages as confirmed by Ki67 expression. Additionally, both SVZ-NSCs sources are capable of self-renewal in addition to NESTIN, SOX2, and GFAP expression. The mortality was measured meager with over 95% viability according to TUNEL and Live/Dead assay results. Eventually, the multipotency of SVZ-NSCs appraised authentic after their differentiation into neurons, astrocytes, and oligodendrocytes. In this study, we proposed a reliable method for SVZ-NSCs in vitro maintenance and identification, which, we believe is a promising cell source for therapeutic approach to recover neurological disorders and injuries condition.